The role of the chemokine receptor CCR5 in HIV infection and disease transmission is well- established; however, the structural mechanisms of CCR5-mediated HIV entry into host cells and inhibition of entry by chemokines remain elusive. This lack of knowledge represents a critical barrier for efforts to combat HIV, hindering rational design of therapeutics targeting the chemokine system and possessing desired HIV inhibition profiles. The long term goal of the applicants' research is to obtain a deep structural understanding of CCR5 interactions with its natural ligands, drugs, and HIV gp120 variants, thus enabling rational design of highly efficient HIV entry inhibitors with reduced susceptibility to development of resistance. The objective of this proposal is to elucidate the structural determinants of the interaction of CCR5 with chemokines in the context of HIV entry inhibition, the interaction of CCR5 with fusogenic gp120 variants facilitating HIV entry, and the tolerance of CCR5 to gp120 sequence diversity in the context of HIV resistance and cellular tropism. The central hypothesis is that the remarkable structural plasticity of CCR5 allows for recognition of diverse ligands and accommodation of drug-resistant HIV strains through a conserved set of binding determinants. This hypothesis has been formulated based on extensive literature review and data obtained in the applicants' laboratories including the first X-ray structure of a receptor:chemokine complex (CXCR4:vMIP-II) that was recently solved as a part of their collaboration. The central hypothesis will be tested by pursuing two Specific Aims: (1) Elucidate the structural determinants of affinity, specificity and antiviral activity of potent CCR5 binding chemokines, and (2) Determine the structural basis of the interaction of gp120 with CCR5 and the mechanisms of resistance and tropism. Specifically, in Aim 1, structure(s) of CCR5 will be solved in complex with variants of the chemokine RANTES that potently inhibit viral entry by two different mechanisms: steric blockade and internalization/sequestration of CCR5 inside cells. Such structures will promote understanding of CCR5 conformations that translate into specific functional responses and enable rational targeting of these conformations with small molecules. In Aim 2, a synergistic computational/experimental approach will be applied to obtain CCR5 complexes with rationally designed gp120 chimeras and full-length gp120. This aim will contribute to a better molecular understanding of how HIV infects cells and achieves resistance and cellular tropism. The overall project is innovative because the structural basis of chemokine and gp120 interactions with CCR5 has not been studied in the context of full length CCR5, and because of novel strategies that heavily integrate computational modeling with experiments to generate stable, crystallizable complexes of these challenging membrane-protein:protein targets. The proposed research is significant because it is expected to vertically advance structural and mechanistic understanding of the chemokine/receptor/HIV system and enable rational targeting of its components.